I kappa b kinase complex as a target for the treatment of huntington&#39;s disease

ABSTRACT

The present invention provides methods and compositions for protecting cells from the toxicity of mutant huntingtin (Htt) protein and for treatment of Huntington&#39;s disease (HD). The methods generally involve administering to cells or a patient an effective amount of an IKK inhibitor. In addition, methods are provided for identifying therapeutics for the treatment of HD.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/218,924, filed Sep. 1, 2005, which claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 60/606,969, filed Sep. 3,2004. The priority application is incorporated herein by reference inits entirety.

STATEMENT REGARDING FEDERALLY SPONSORED R&D

This invention was made with government support under grantNS045165-01A1 awarded by the National Institutes of NeurologicalDisorders and Stroke. The government has certain rights in theinvention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to targets for treatingHuntington's disease, and more specifically to methods and compositionsfor protecting cells from the toxicity of mutant Htt and treatingHuntington's disease in an animal.

2. Description of the Related Art

Huntington's disease (HD) is a fatal autosomal dominantneurodegenerative disorder that is caused by the expansion of CAGrepeats in exon 1 (HDx1) of huntingtin gene (Reddy et al. TrendsNeurosci. 22:248-255 (1999)). The huntingtin gene is known and is thesubject of U.S. Pat. No. 5,693,757, incorporated herein by reference.Expanded CAG repeats (40 and above), which form an abnormalpolyglutamine (polyQ) stretch in the huntingtin (Htt) protein, result ina gain of toxic function and induce death in subpopulations of neuronsin the striatum and cortex (Zoghbi et al. Annu. Rev. Neurosci.23:217-247 (2000); Tobin et al. Trends Cell Biol. 10:531-536 (2000)).Neuronal death in HD has been attributed not only to polyQ toxicity, butalso to activation of caspases, transcriptional dysregulation, andsequestration/inactivation of wild-type Htt and other important cellularfactors. As disclosed below, the NF-κB pathway, which plays a centralrole in cell death and survival, is effected by mutant Htt.

NF-κB is sequestered in the cytoplasm by a family of inhibitory proteins(Iκ-Bs) (Ghosh et al., Annu Rev Immunol 16:225-260 (1998)). Iκ-Bs arephosphorylated by a signal-activated kinase complex known as I-κB kinase(IKK) (Ghosh and Karin, Cell [Suppl] 109:81-96 (2002)). This complexcontains two catalytic subunits, IKKα and IKKβ, and a regulatory module,IKKγ (Karin and Lin, Nat Immunol 3:221-227 (2002)). Phosphorylated Iκ-Bsare ubiquitinated by an F-box E3-ligase, β-transducin repeat-containingprotein (β-TrCP) (Spencer et al., Genes Dev 13:284-294 (1999) andsubsequently degraded by proteosomes. Liberated NF-κB can bind DNA andpromote gene expression (Pahl, Oncogene 18:6853-6866 (1999)).

SUMMARY OF THE INVENTION

In one aspect, the invention relates to methods for protecting cells,preferably in a mammal, from the toxicity of mutant Htt by blockingactivation of IKK. This may be done by administering to the mammal aneffective amount of an IKK inhibitor. In another aspect, the inventionrelates to methods for treatment of HD by administering an effectiveamount of an IKK inhibitor to a patient. In some embodiments the IKKinhibitor blocks activation of IKK.

Preferably, the IKK inhibitor acts directly on IKK. However, in someembodiments the IKK inhibitor may inhibit the activity of a molecule ina downstream IKK dependent pathway, such as βTrCP. In other embodimentsthe inhibitor may block the interaction between IKK and mutant Htt.Preferred molecules useful for inhibiting IKK include small moleculeinhibitors, antibodies, dominant negative forms of IKK, such as DN-IKKγ,and dominant negative forms of the E3-ubiquitin ligase βTrCP, such asΔF-βTrCP. In some embodiments the IKK inhibitor binds to one or moresubunits of IKK, such as IKKβ. The IKK inhibitor may block theinteraction of IKK subunits, such as the interaction of IKKγ with IKKβor IKKα. In another embodiment, the IKK inhibitor blocks IKKβactivation.

In some embodiments, the IKK inhibitor is a small molecule selected fromthe group consisting of herbimycin, NEMO binding peptide, sodiumsalicylate, retinoid-related compounds, and cyclopentenoneprostaglandins. In another embodiment, the IKK inhibitor is an antibody.In still other embodiments, the IKK inhibitor is an antibody, such as amonoclonal antibody that inhibits IKK activity.

In another aspect, the methods of the invention involve the treatment ofan individual, more preferably a mammal and even more preferably a humanhaving, suspected of having and/or at risk of developing HD byadministering a therapeutically effective amount of an IKK inhibitor tothe individual. In preferred embodiments the IKK inhibitor compositionsof the methods are preferably delivered intracranially, for example, byinjection directly into brain tissue or by injection into thecerebrospinal fluid. However, in other embodiments, for example wherethe IKK inhibitor is able to cross the blood brain barrier, the IKKinhibitor is administered peripherally.

In another aspect, the present invention provides an article ofmanufacture that comprises a container, a pharmaceutical compositioncomprising an IKK inhibitor within the container and instructions toadminister the pharmaceutical composition at a dose which is betweenabout 0.01 μg/kg and about 1 mg/kg.

In another aspect, the present invention provides methods of screeningfor an IKK inhibitor useful for protecting cells, preferably in amammal, from the toxicity of mutant Htt. Preferably the IKK inhibitorblocks activation of IKK. IKK inhibitors that are identified can be usedfor treating HD.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows mutant Htt activates NF-κB-dependent gene expression. A, isa graph illustrating that PC12 cells expressing mutant HDx1 showelevated NF-κB activity. The graph shows luciferase units from a plasmidwith a minimal promoter containing NF-κB binding sites. B, is a graphillustrating that expression of WT or mutant HDx1 does not influenceluciferase expression from a control plasmid, which lacks an NF-κBenhancer element. C, Is a western blot showing that NF-κB activation bymutant Htt includes degradation of Iκ-Bα. PC12 cells were treated withecdysone to induce HDx1 for the indicated time and examined by Westernblotting with anti-Iκ-Bα antibody. D, is a graph illustrating thatfull-length mutant Htt enhances IL-1β-mediated NF-κB activation.Striatal cells from WT and HD KI mice were transfected with NF-κB or thecontrol reporter and the indicated plasmids.

FIG. 2 shows that mutant Htt promotes activation of nuclear localizationof NF-κB. A, is a graph illustrating binding of nuclear proteins fromcontrol and IL-1β-treated striatal cells from WT or mutant Htt KI miceto the oligonucleotide recognized by NF-κB. B, is a graph showing thatthe nuclear localization of the NF-κB p65 subunit is elevated in HDtransgenic brain. C, is a micrograph of nuclear p65 staining showing theaverage percentage of positive neurons per microscopic field from 16brain sections of four animals each for WT and HD mice.

FIG. 3 shows that mutant HDx1 activates the IKK complex. A, is a blotIKK complexes isolated from PC12 cells expressing mutant HDx1 haveelevated kinase activity. Similar results were obtained when IKKcomplexes were isolated from striatal or cortical extracts of HDtransgenic R6/2 mice (B) or striatal HD knock-in mice (C). D, is aWestern blot showing that mutant HDx1 coprecipitates with endogenous IKKcomplex. E, is a Western blot showing that mutant Httcoimmunoprecipitates with IKK complexes isolated from R6/2 striatalextracts.

FIG. 4 shows mutant Htt directly interacts with IKKγ. A, shows severalgells demonstrating that full-length (F) and C-terminal-truncated IKKγ(DC) bind to mutant HDx1 (51polyQ). B, shows a gel confirming thatbinding of IKKγ to mutant HDx1 requires the Htt polyQ and polyP domains.C, is a graph illustrating that expression of anti-HDx1 recombinantantibodies, which block binding of IKKγ to Htt, reduces NF-κB-dependentgene expression.

FIG. 5 shows DN-IKKγ reduces the toxicity of mutant HDx1. A, IKKγpromotes aggregation and nuclear localization of Htt. B, DN-IKKγexpression reduces the toxicity of mutant HDx1. C, is a graphillustrating that DN-IKKγ blocks HDx1-induced NF-κB activation.

FIG. 6 shows DN-IKKγ protects striatal MSNs against mutant HDx1. A, is aseries of micrographs showing that living MSNs can be monitored formorphology and Htt expression. B, is a graph illustrating thatexpression of DN-IKKγ is neuroprotective for MSNs in brain slices.

FIG. 7 shows ΔF-βTrCP reduces the toxicity of mutant Htt. A, is a graphillustrating that NF-κB activation by mutant HDx1 is blocked byΔF-βTrCP. B, is a series of micrographs showing that mutant HDx1 andΔF-βTrCP are colocalized in living HEK-293 cells. C, is a graphillustrating that apoptotic bodies in transfected HEK-293 cells arereduced by ΔF-βTrCP. D, is a graph illustrating that expression ofΔF-βTrCP reduces the toxicity of mutant HDx1 in MSNs.

DETAILED DESCRIPTION

One embodiment of the present invention is a treatment for Huntington'sdisease (HD). This embodiment is based, in part, on the discovery thatinhibition of I-κB kinase (IKK) activity can protect against thetoxicity associated with mutant huntingtin protein (Khoshnan et al.,Journal of Neuroscience 24(37) (2004)), expressly incorporated herein byreference) which leads to manifestation of HD in humans. In someembodiments cells are protected from the toxicity of mutant Htt bycontacting the cells with an IKK inhibitor.

In other embodiments, patients suffering from Huntington's disease (HD)are treated by blocking IKK activation in vivo, as described in moredetail below. Preferred molecules useful for inhibiting IKK includesmall organic molecules, peptides and antibodies. Compounds that inhibitIKK activity and are thus suitable for use in the methods of the presentinvention include, without limitation, herbimycin, NEMO bindingpeptides, sodium salicylate, retinoid-related compounds, andcyclopentenone prostaglandins. Herbimycin binds IKKβ and blocksactivation of IKK. NEMO binding peptides block the interaction of IKKγwith IKKβ and IKKα, thereby blocking IKK activation. Sodium salicylate,retinoid-related compounds and cyclopentenone prostaglandins alsoinhibitors of IKKβ activation.

In other embodiments, compounds that can be used to treat HD areidentified by screening compounds for their ability to inhibit IKKactivity. Compounds that can be screened include, without limitation,small organic molecules, peptides and antibodies.

These and other embodiments are described in more detail below.

DEFINITIONS

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. See, e.g. Singleton et al.,Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley &Sons (New York, N.Y. 1994); Sambrook et al., Molecular Cloning, ALaboratory Manual, Cold Springs Harbor Press (Cold Springs Harbor, N.Y.1989). For purposes of the present invention, the following terms aredefined below.

“Huntingtin” and “Htt” refer broadly to the huntingtin gene and theprotein encoded by the huntingtin gene, including mutant and variantforms as well as native forms. “Variants” are biologically activepolypeptides having an amino acid sequence which differs from thesequence of a native sequence polypeptide. Native sequence humanhuntingtin protein is described, for example, by The Huntington'sDisease Collaborative Research Group in Cell 72:971-983 (1993) as wellas in Li et al. Nature 378:398-402 (1995) and WO 02/29408. The number ofpolyglutamine repeats in native huntingtin protein is known to vary,from about 13 to about 36 glutamine residues in the polyQ region ofnative human protein. Native sequence murine Htt is described, forexample, in Lin et al. Hum. Mol. Genet. 3 (1), 85-92 (1994) andtypically comprises about 7 glutamine residues in the polyQ region.Particular variants of the huntingtin gene will comprise differentnumbers of CAG repeats, resulting in variation in the polyglutamineregion of the huntingtin protein.

“Mutant huntingtin protein” refers to huntingtin protein which differsin some respect from the native sequence huntingtin protein. Typically,mutant huntingtin will comprise an expanded polyglutamine or polyprolineregion compared to the native form. A preferred mutant huntingtinprotein has an expanded polyglutamine region of 40 or more glutamineresidues.

“IKK” refers broadly to the Iκ-B kinase complex. The Iκ-B kinase complexcomprises two catalytic subunits IKKα and IKKβ and a regulatory subunitIKKγ.

The term “IKK inhibitor” is used in the broadest sense and includes anymolecule that partially or fully blocks, inhibits or neutralizes abiological activity mediated by IKK, preferably by preventing theactivation of IKK. Preferred IKK inhibitors act directly on one or moresubunits of IKK, for example by binding to one or more subunits of IKK.However, in other embodiments the IKK inhibitors may prevent IKK frominteracting with a substrate, such as I-κB and/or may act on moleculesin an IKK signaling pathway, preferably downstream from IKK. In stillother embodiments the IKK inhibitors may modulate the level of IKK geneexpression or otherwise reduce the levels of IKK in affected cells.

The ability of a molecule to inhibit IKK activation can be measuredusing assays that are well known in the art. For example and withoutlimitation, IKK inhibitors can be identified using immune complex kinaseassays and gene reporter assays. Briefly, in an immune complex kinaseassay, immunoprecipitated IKK complexes are examined for the ability tophosphorylate GST-Iκ-Bα in vitro. For example, IKK complexes can beimmunoprecipitated from cleared striatal extracts from animals or cellstreated with the putative IKK inhibitor by incubation with a mouseanti-IKKα antibody (Santa Cruz Biotechnology) coupled to protein-A beadsand rocked for 3 hr at 4° C. Beads are washed, and IKK activity can beevaluated in vitro with 1 μg of purified GST-Iκ-Bα (N-terminal 61 aminoacids) in the presence of 10 μCi of [³²P]γ-ATP for 30 min at 30° C.Products are examined by SDS-PAGE followed by autoradiography.

Gene reporter assays can be used to measure downstream effects of IKK,such as NF-κB activation. For example, a plasmid based reporter,pNF-κB-luciferase, with five enhancer elements and a control plasmidwithout NF-κB binding sites, pCIS-CK-luciferase, can be used to verifyinhibition of NF-κB activity in cells treated with the putativeinhibitor (Stratagene, La Jolla, Calif.). The skilled artisan will beable to select the appropriate assays and reaction conditions based onthe particular circumstances.

Furthermore, the term “IKK inhibitor” includes any molecule that mimicsa biological activity mediated by an IKK subunit and specificallychanges the function or expression of IKK, or the efficiency ofsignaling through IKK, thereby inhibiting an already existing biologicalactivity or triggering a new biological activity.

“Biological property” or “biological activity” is a biological functioncaused by an IKK, an IKK inhibitor, or other compound of the invention.Biological properties of IKKs include the phosphorylation of I-κB andactivation of NF-κB dependent pathways. With regard to the IKKinhibitors, biological activity refers, in part, to the ability toinhibit activation of IKK. Other preferred biological activities of IKKinhibitors include prevention of cell death or apoptosis, inhibition ofNF-κB dependent gene transcription and the ability to regulate andpreferably reduce or eliminate the toxic effects of mutant huntingtinprotein that are associated with neurodegenerative disease.

The term “therapeutically effective amount” or “therapeuticallyeffective dose” refers to an amount effective to treat a disease ordisorder in a mammal. In the case of HD, the therapeutically effectiveamount of an IKK inhibitor prevents cell death associated with mutantHtt and/or reduces one or more of the symptoms of HD. Thetherapeutically effective dose may be a single dose, or may comprisemultiple doses given over a period of time.

The term “antibody” is used herein in the broadest sense andspecifically covers human, non-human (e.g. murine) and humanizedmonoclonal antibodies, including full length monoclonal antibodies,polyclonal antibodies, multi-specific antibodies, and antibodyfragments, including intrabodies, so long as they exhibit a desiredbiological activity. Antibodies exhibit binding specificity to aspecific antigen.

An “individual” is a vertebrate, preferably a mammal, more preferably ahuman.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, horses, cats, cows, etc. Preferably, themammal herein is human.

As used herein, “treatment” is a clinical intervention made in responseto and in anticipation of a disease, disorder or physiological conditionmanifested by a patient, particularly Huntington's disease. The aim oftreatment includes the alleviation or prevention of symptoms, slowing orstopping the progression or worsening of a disease, disorder, orcondition and/or the remission of the disease, disorder or condition.“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures. Those in need of treatment include those alreadyaffected by a disease or disorder or undesired physiological conditionas well as those in which the disease or disorder or undesiredphysiological condition is to be prevented.

In the methods of the present invention, the term “control” andgrammatical variants thereof, are used to refer to the prevention,partial or complete inhibition, reduction, delay or slowing down of anunwanted event, such as the presence or onset of Huntington's disease.

IKK Inhibitors

In some embodiments of the invention, cells are protected from thetoxicity of mutant Htt protein by administering an effective amount ofan IKK inhibitor. In other embodiments HD is treated by administering anIKK inhibitor to a patient. A variety of molecules are known to inhibitIKK activity and can be used to reduce toxicity of Htt in cells or totreat a patient having or expected to develop HD. The method by whichIKK is inhibited is not limited in any way, and may be, for example, byblocking IKK subunit interaction. In addition, the present inventionprovides methods for screening for molecules useful for reducingtoxicity of Htt in cells and for treating HD.

In some embodiments, the IKK inhibitor preferably interacts with IKK andblocks activation. However, in other embodiments the IKK inhibitor mayinterfere with the interaction of IKK with a binding partner orsubstrate or may interfere with IKK gene expression or otherwisemodulate the levels of IKK in the body. In still other embodiments theIKK inhibitor may interact with a molecule that is in an IKK dependentpathway, preferably downstream from IKK.

The type of IKK inhibitor is not limited in any way. Preferred IKKinhibitors include, for example, small molecule inhibitors, antibodies,proteins, etc. In one embodiment, the IKK inhibitor is a small moleculethat binds to IKKβ, for example a Src tyrosine kinase inhibitor such asherbimycin. Herbimycin A abrogates NF-κB activation by interacting withthe IKKβ subunit (Ogino et al., Mol. Pharmacol. 65(6):1344-51 (2004)).In other embodiments the IKK inhibitor is a compound that blocksinteraction of IKK subunits, such as the interaction of IKKγ with IKKβand IKKα. An example of such a compound is a NEMO (IKKγ) binding peptide(see, for example, Dai et al. J. Biol. Chem. 279(36):37219 (2004);Eijiro, J., et al. Nat. Med. 10(6):617 (2004); Siegmund, D., et al. J.Biol. Chem. 276:43708 (2001); May, M. J., et al. Science 289:1550(2000); and Li, Q., et al. Science 284:1999 (1999), incorporated byreference herein). A NEMO binding peptide that is used in someembodiments is available from Calbiochem (Cat. No. 480025). In anotherembodiment, the IKK inhibitor is a compound that blocks IKKβ activation,for example, sodium salicylate. In another embodiment, the IKK inhibitoris a retinoid-related compound or a cyclopentenone prostaglandin.

In other embodiments the inhibitor is a compound that blocks theinteraction of IKK with mutant Htt. In some such embodiments thecompound that blocks the interaction of IKK with mutant Htt is a peptideor small organic molecule. Preferably the compound binds IKK.

Antibodies that can block activation of IKK are also suitable for use inmethods of the present invention. Preferred antibodies bind to one ormore subunits of the IKK complex. For example, antibodies that aretargeted to one or more subunits of IKK and prevent interaction of thesubunits can be used. In other embodiments an antibody that prevents IKKfrom phosphorylating I-κB can be used. In still other embodiments ananti-IKK antibody prevents interaction of IKK and mutant Htt. Theantibodies are not limited in any way, but are preferably monoclonalantibodies, more preferably human or humanized monoclonal antibodies.Antibodies to IKK can be prepared using methods that are well known inthe art and inhibitory antibodies can be identified using the methodsdescribed herein.

Other proteins that can block activation of IKK or otherwise inhibit IKKactivity are also suitable for use in the methods of the presentinvention. For example, expression of DN-IKKγ or ΔF-βTrCP reduces thetoxicity of mutant Htt in cell culture and protects striatal MSNs in abrain slice model of HD. DN-IKKγ lacks the binding domain essential forinteraction with IKKα and -β and thus inhibits IKK activity (May et al.,Science 289:1550-1554 (2000); Poyet et al., J Biol Chem 275:37966-37977(2000)). DN-IKKγ also blocks basal and NGF-induced NF-κB-dependent geneexpression by mutant HDx1 in inducible PC12 cells (FIG. 5C). TheE3-ubiquitin ligase βTrCP specifically promotes degradation of Iκ-Bα(Spencer et al., Genes Dev 13:284-294 (1999)). A dominant-negative formof βTrCP, ΔF-βTrCP, blocks degradation of phosphorylated Iκ-Bs andabolishes basal and mutant HDx1-induced NF-κB activity (FIG. 7A). Thus,DN-IKKγ, and ΔF-βTrCP can be used to block IKK activation for purposesof the present invention.

In some embodiments proteins that are able to block IKK activation areprovided directly to the patient, such as by injection. In otherembodiments nucleic acids encoding the proteins are obtained andinserted into appropriate expression vectors. Cells that may be subjectto mutant Htt toxicity may then be transfected with the expressionvector, such that the protein is expressed in the cells. Methods forsuch genetic therapies are known in the art and can be adapted by theskilled artisan as necessary. This includes both gene therapy where alasting effect is achieved by a single treatment, and gene therapy wherethe increased expression is transient. Selective expression of IKKinhibitory proteins in appropriate cells may be achieved by usingvectors with tissue specific or inducible promoters or by producinglocalized infection with replication defective viruses, or by any othermethod known in the art.

Compositions Comprising IKK Inhibitors

One embodiment of the invention is a method of treatment involvingadministration of an effective amount of a composition comprising an IKKinhibitor. In some embodiments, the composition comprises an IKKinhibitor that is a small molecule or peptide, for example an IKKinhibitor selected from the group consisting of herbimycin, NEMO bindingpeptide, sodium salicylate, retinoid-related compounds, andcyclopentenone prostaglandins. In other embodiments the compositioncomprises an IKK inhibitor that is an antibody or other polypeptide,such as a human or humanized anti-IKK monoclonal antibody.

In pharmaceutical dosage forms, the IKK inhibitors may be used alone orin appropriate association, as well as in combination with otherpharmaceutically active or inactive compounds. The IKK inhibitors can beformulated into pharmaceutical compositions containing a single IKKinhibitor or a combination of two or more IKK inhibitors. For example, apharmaceutical composition can contain two or more different IKKinhibitors. In one embodiment, the pharmaceutical composition containstwo or more different IKK inhibitors having the same mode of action. Forexample, both IKK inhibitors may disrupt subunit interaction. In anotherembodiment, the pharmaceutical composition contains two or more IKKinhibitors having different methods of action. For example, oneinhibitor may disrupt subunit interaction while a different inhibitorprevents IKK from phosphorylating I-κB.

The IKK inhibitors can be formulated into pharmaceutical compositions bycombination with appropriate, pharmaceutically acceptable carriers ordiluents (Remington, The Science and Practice of Pharmacy, 19^(th)Edition, Alfonso, R., ed., Mack Publishing Co., Easton, Pa. (1995)), andmay be formulated into preparations in solid, semi-solid, liquid orgaseous forms, such as tablets, capsules, powders, granules, ointments,solutions, suppositories, injections, inhalants and aerosols dependingon the particular circumstances.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are readily available. Moreover, pharmaceuticallyacceptable auxiliary substances, such as pH adjusting and bufferingagents, antioxidants, low molecular weight (less than about 10 residues)polypeptides, tonicity adjusting agents, stabilizers, wetting agents andthe like, are readily available. “Carriers” when used herein refers topharmaceutically acceptable carriers, excipients or stabilizers whichare nontoxic to the cell or mammal being exposed to the carrier at thedosages and concentrations used.

The IKK inhibitors to be used for in vivo administration are preferablysterile. The sterility may be accomplished by any method known in theart, such as by filtration using sterile filtration membranes, prior toor following lyophilization and reconstitution. In some embodiments theIKK inhibitors are available commercially in sterile form.

The IKK inhibitor compositions may be placed into a container with asterile access port, for example, an intravenous solution bag or vialhaving a stopper pierceable by a hypodermic injection needle.

The inhibitors can be formulated into preparations for injection bydissolving, suspending or emulsifying them in an aqueous or nonaqueoussolvent, such as vegetable or other similar oils, synthetic aliphaticacid glycerides, esters of higher aliphatic acids or propylene glycol;and if desired, with conventional additives such as solubilizers,isotonic agents, suspending agents, emulsifying agents, stabilizers andpreservatives.

The compounds can be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection can be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionscan take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and can contain formulatory agents such as suspending,stabilizing or dispersing agents. Alternatively, the active ingredientcan be in powder form for constitution with a suitable vehicle, e.g.,sterile pyrogen-free water, before use. The compounds can also beformulated in rectal compositions such as suppositories or retentionenemas, e.g., containing conventional suppository bases such as cocoabutter or other glycerides.

In addition to the formulations described previously, the compounds canalso be formulated as a depot preparation. Such long acting formulationscan be administered by implantation (for example, subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds can be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

For oral preparations, the IKK inhibitors can be combined withappropriate additives to make tablets, powders, granules or capsules.For example, the IKK inhibitor can be combined with conventionaladditives such as lactose, mannitol, corn starch or potato starch; withbinders, such as crystalline cellulose, cellulose derivatives, acacia,corn starch or gelatins; with disintegrators, such as corn starch,potato starch or sodium carboxymethylcellulose; with lubricants, such astalc or magnesium stearate; and if desired, with diluents, bufferingagents, moistening agents, preservatives and flavoring agents. Liquidpreparations for oral administration can take the form of, for example,solutions, syrups or suspensions, or they can be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Preparations for oral administration can be suitably formulated togive controlled release of the active compound.

IKK inhibitors can also be aerosolized or otherwise prepared foradministration by inhalation. For example a fluorocarbon formulation anda metered dose inhaler, or inhaled as a lyophilized and milled powder.For administration by inhalation, the IKK inhibitors can be utilized inaerosol formulation to be administered via inhalation. The compounds ofthe present invention can be formulated into pressurized acceptablepropellants such as dichlorodifluoromethane, propane, nitrogen and thelike.

If an IKK inhibitor is coadministered with another IKK inhibitor, orwith another agent having similar biological activity, the differentactive ingredients may be formulated together in an appropriate carriervehicle to form a pharmaceutical composition. Alternatively, the IKKinhibitors can be formulated separately and administered simultaneouslyor in sequence.

The compositions can, if desired, be presented in a pack or dispenserdevice that can contain one or more unit dosage forms containing theactive ingredient. The pack can for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device can beaccompanied by instructions for administration.

Methods of Treatment

Is some embodiments, an individual suffering from or at risk of HD istreated (including prevention) by administering a composition comprisingone or more IKK inhibitors at a therapeutically effective dose. Asdiscussed above, treatment can include an amelioration of the symptomsassociated with the pathological condition afflicting the host, whereamelioration is used in a broad sense to refer to at least a reductionin the magnitude of a parameter, e.g. symptom, associated with thepathological condition being treated, such as neuronal cell death. Assuch, treatment includes situations where the pathological condition, orat least symptoms associated therewith, are completely inhibited, e.g.prevented from happening, or stopped, e.g. terminated, such that thehost no longer suffers from the pathological condition, or at least thesymptoms that characterize the pathological condition. However,treatment can also be delay or slowing of disease progression,amelioration or palliation of the disease state, and remission (whetherpartial or total), whether detectable or undetectable.

A variety of individuals are treatable. Generally such individuals aremammals, where the term is used broadly to describe organisms which arewithin the class mammalia, including the orders carnivore (e.g., dogsand cats), rodentia (e.g., mice, guinea pigs, and rats), and primates(e.g., humans, chimpanzees, and monkeys). In preferred embodiments, theindividuals are humans.

The IKK inhibitors may be administered using any convenient protocolcapable of resulting in the desired therapeutic activity. A specificprotocol can readily be determined by a skilled practitioner withoutundue experimentation based on the particular circumstances. Thus, theIKK inhibitor can be incorporated into a variety of formulations fortherapeutic administration, as discussed above, depending on theprotocol adapted by the supervising clinician.

Each dosage for human and animal subjects preferably contains apredetermined quantity of one or more IKK inhibitors calculated in anamount sufficient to produce the desired effect, in association with apharmaceutically acceptable diluent, carrier or vehicle. Again, theactual dosage forms will depend on the particular compound employed, theeffect to be achieved, and the pharmacodynamics associated with eachcompound in the host.

Administration of the IKK inhibitors can be achieved in various ways,including intracranial, for example injection directly into the braintissue or into the cerebrospinal fluid, oral, buccal, rectal,parenteral, intraperitoneal, intradermal, transdermal, intracheal,intracerebral, etc., administration. The IKK inhibitors may beadministered alone or in combination with one or more additionaltherapeutic agents. Administration “in combination with” one or morefurther therapeutic agents includes both simultaneous (at the same time)and consecutive administration in any order.

Administration may be chronic or intermittent, as deemed appropriate bythe supervising practitioner, particularly in view of any change in thedisease state or any undesirable side effects. “Chronic” administrationrefers to administration of the IKK inhibitor in a continuous mannerwhile “intermittent” administration refers to treatment that is not donewithout interruption.

Combinations of IKK inhibitors for simultaneous administration are usedin some embodiments. For example, two or more different IKK inhibitorsmay be administered in combination.

In a particular embodiment, IKK inhibitors of the invention areadministered by intracranial injection. The injection will typically bedirectly into affected brain regions or into the cerebrospinal fluid.

An effective amount of an IKK inhibitor to be employed therapeuticallywill depend, for example, upon the therapeutic objectives, the route ofadministration, the nature of the IKK inhibitor, and the condition ofthe patient. Accordingly, it will be necessary for the therapist totiter the dosage and modify the route of administration as required toobtain the optimal therapeutic effect. A typical daily dosage mightrange from about 0.01 μg/kg to up to 1 mg/kg or more, depending on thefactors mentioned above. Preferably, a typical daily dosage ranges fromabout 1 μg/kg to about 100 μg/kg. Typically, the clinician willadminister an IKK inhibitor until a dosage is reached that provides thebest clinical outcome. The progress of this therapy is easily monitoredby conventional assays.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Compounds exhibiting large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects can be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize undesired side effects.

Screening Assays for IKK Inhibitors

In other embodiments, therapeutics useful in treating HD are identifiedby screening for compounds that inhibit IKK activity. Screening assaysare well known in the art and can readily be adapted to identify IKKinhibitors. As discussed above, such IKK inhibitors may includecompounds that interact with (e.g., bind to) IKK, compounds thatinterfere with the interaction of IKK with its binding partners, cognateor substrate, and compounds that modulate IKK gene expression, such ascompounds that modulate the level of IKKγ gene expression, or otherwisemodulate the levels of IKK in the body. Assays may additionally beutilized which identify compounds that bind to IKK gene regulatorysequences (e.g., promoter sequences) and, consequently, may modulate IKKgene expression. See, Platt, K. A., 1994, J. Biol. Chem.269:28558-28562, which is incorporated herein by reference in itsentirety.

The compounds which may be screened in accordance with the inventioninclude, but are not limited to small molecules, peptides, antibodiesand fragments thereof, and other organic compounds (e.g.,peptidomimetics). The compounds may include, but are not limited to,soluble peptides, including members of random peptide libraries; (see,e.g., Lam, K. S. et al., 1991, Nature 354:82-84; Houghten, R. et al.,1991, Nature 354:84-86), and combinatorial chemistry-derived molecularlibraries made of D- and/or L-configuration amino acids, phosphopeptides(including, but not limited to members of random or partiallydegenerate, directed phosphopeptide libraries; see, e.g., Songyang, Z.et al., 1993, Cell 72:767-778), antibodies (including, but not limitedto, polyclonal, monoclonal, humanized, anti-idiotypic, chimeric orsingle chain antibodies, and FAb, F(abN)₂ and FAb expression libraryfragments, and epitope-binding fragments thereof), and small organic orinorganic molecules, including libraries thereof.

Other compounds which can be screened in accordance with the inventioninclude, but are not limited to small organic molecules, including butnot limited to those that are able to cross the blood-brain barrier.

Libraries of known compounds, including natural products or syntheticchemicals, and biologically active materials, including proteins, may bescreened for compounds which are inhibitors of IKK. One method whichdetects protein interactions in vivo and can be used to identify IKKinhibitors, the two-hybrid system, is described in detail forillustration only and not by way of limitation. One version of thissystem has been described (Chien et al., 1991, Proc. Natl. Acad. Sci.USA, 88:9578-9582) and is commercially available from Clontech (PaloAlto, Calif.). The two-hybrid system may be adapted for screening forsmall molecule IKK inhibitors.

Briefly, in the two-hybrid system, plasmids are constructed that encodetwo hybrid proteins: one plasmid consists of nucleotides encoding theDNA-binding domain of a transcription activator protein fused to anucleotide sequence encoding, for example, an IKK subunit, such as IKKγ,or a polypeptide, peptide, or fusion protein therefrom, and the otherplasmid consists of nucleotides encoding the transcription activatorprotein's activation domain fused to a cDNA encoding an unknown proteinwhich has been recombined into this plasmid as part of a cDNA library.The DNA-binding domain fusion plasmid and the cDNA library aretransformed into a strain of the yeast Saccharomyces cerevisiae thatcontains a reporter gene (e.g., HBS or lacZ) whose regulatory regioncontains the transcription activator's binding site. Either hybridprotein alone cannot activate transcription of the reporter gene: theDNA-binding domain hybrid cannot because it does not provide activationfunction and the activation domain hybrid cannot because it cannotlocalize to the activator's binding sites. Interaction of the two hybridproteins reconstitutes the functional activator protein and results inexpression of the reporter gene, which is detected by an assay for thereporter gene product.

The two-hybrid system may be adapted to screen for and identify smallmolecule IKK inhibitors that, for example, can disrupt the interactionof IKK subunits or the interaction of IKK and mutant Htt. By way ofexample, and not by way of limitation, an IKK yeast reporter strain canbe generated by cotransforming a plasmid encoding an IKK subunit, suchas IKKβ, gene product fused to a DNA-activation domain and a plasmidencoding a hybrid of an IKKγ gene product fused to the DNA-bindingdomain into a yeast reporter strain. The resulting IKK yeast reporterstrain is useful for screening libraries, such as small moleculelibraries, for IKK inhibitors. Small molecules capable of disrupting theIKK subunit interaction inhibit expression of the lacZ reporter.

The two-hybrid system or related methodology may also be used to screenactivation domain libraries for proteins that interact with the “bait”gene product. By way of example, and not by way of limitation, an IKKsubunit, such as IKKγ, can be used as the bait gene product. Totalgenomic or cDNA sequences are fused to the DNA encoding an activationdomain. This library and a plasmid encoding a hybrid of a bait IKKγ geneproduct fused to the DNA-binding domain are cotransformed into a yeastreporter strain, and the resulting transformants are screened for thosethat express the reporter gene. For example, and not by way oflimitation, a bait IKKγ gene sequence, e.g., the genes open readingframe, can be cloned into a vector such that it is translationally fusedto the DNA encoding the DNA-binding domain of the GAL4 protein. Thepositive clones that display positive interaction are identified and thelibrary plasmids responsible for reporter gene expression are isolated.DNA sequencing is then used to identify the proteins encoded by thelibrary plasmids.

A cDNA library of the cell line from which proteins that interact withthe bait IKKγ gene product are to be detected can be made using methodsroutinely practiced in the art. According to the particular systemdescribed herein, for example, the cDNA fragments can be inserted into avector such that they are translationally fused to the transcriptionalactivation domain of GAL4. This library can be co-transformed along withthe bait IKKγ gene-GAL4 fusion plasmid into a yeast strain whichcontains a lacZ gene driven by a promoter which contains a GAL4activation sequence. A cDNA encoded protein, fused to GAL4transcriptional activation domain, that interacts with the bait IKKγgene product will reconstitute an active GALA protein and thereby driveexpression. Colonies which drive expression can be detected by methodsroutine in the art. The cDNA can then be purified from these strains,and used to produce and isolate the bait IKKγ gene-interacting proteinusing techniques routinely practiced in the art.

Small molecules may also have the ability to act as IKK inhibitors andthus may be screened for such activity. Small molecules preferably havea molecular weight of less than 10 kD, more preferably less than 5 kDand even more preferably less than 2 kD. Such small molecules mayinclude naturally occurring small molecules, synthetic organic orinorganic compounds, peptides and peptide mimetics. However, smallmolecules in the present invention are not limited to these forms.Extensive libraries of small molecules are commercially available and awide variety of assays are well known in the art to screen thesemolecules for the desired activity.

Candidate IKK inhibitor small molecules are preferably first identifiedin an assay that allows for the rapid identification of potentialinhibitors. An example of such an assay is a binding assay wherein theability of the candidate molecule to bind to IKK is measured. Suchassays are well known in the art. Candidate molecules that areidentified by their ability to bind to IKK may then be tested for theirability to inhibit one or more biological activities if IKK. Thistesting may include, for example, an immune complex kinase assay or genereporter assay as described above. Compounds that appear to inhibit IKKactivity can then be tested for their ability to prevent Htt mediatedtoxicity, preferably in a cell based assay, for example as described inExample 5 below. Compounds that are able to prevent or reduce Httmediated toxicity are identified as therapeutics for treating HD.

Articles of Manufacture

In another aspect of the invention, articles of manufacture containingmaterials useful for the treatment of HD are provided. The articles ofmanufacture preferably comprise a container and a label or packageinsert on or associated with the container. Suitable containers include,for example, bottles, vials, syringes, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition which is effective for treating HD and may have asterile access port (for example the container may be an intravenoussolution bag or a vial having a stopper pierceable by a hypodermicinjection needle). At least one active agent in the composition is anIKK inhibitor. The label or package insert indicates that thecomposition is used for treating HD or a related disorder. In addition,the label or package insert may indicate that the patient to be treatedis one who suffers from, or is likely to suffer from or developHuntington's disease or a related disorder.

Alternatively, or additionally, the article of manufacture may furthercomprise a second (or third) container comprising one or more of anothertherapeutic agent, and a pharmaceutically-acceptable buffer, such asbacteriostatic water for injection (BWFI), phosphate-buffered saline,Ringer's solution or dextrose solution. It may further include othermaterials desirable from a commercial end user standpoint, includingother buffers, diluents, filters, needles, and syringes.

EXAMPLES

The following examples, including the experiments conducted and resultsachieved are provided for illustrative purposes only and are not to beconstrued as limiting upon the present invention.

As demonstrated in the Examples below, mutant Htt can bind IKKγ,activate the IKK complex, and elevate NF-κB-dependent gene expression.Toxicity associated with this mutant Htt activity can be significantlyreduced by inhibition of IKK activity.

Example 1 demonstrates that cultured cells expressing mutant Htt andstriatal cells from HD transgenic mice have elevated NF-κB activity.Furthermore, NF-κB was found to be concentrated in the nucleus ofneurons in the brains of HD transgenic mice. Mutant Htt was found toactivate the IKK complex both in cell culture and in mouse models of HD(FIG. 3), as shown in Example 2. Example 3 demonstrates that activationof IKK is mediated by direct interaction with mutant Htt. In particular,the expanded polyglutamine stretch and adjacent proline-rich motifs inmutant Htt interact with IKKγ, a regulatory subunit of IKK. Optimal HDx1binding to the IKK complex was found to require interaction with thefirst N-terminal 134 amino acids of IKKγ (FIG. 4), a domain that isessential for interaction with IKKβ and IKKα, two catalytic units of theIKK complex (May et al., Science 289:1550-1554 (2000)). Interaction ofHtt with IKKγ required polyQ expansion as well as the proline-richmotifs of HDx1.

As shown in Example 4, activation of IKK was found to mediate thetoxicity of mutant Htt. Expression of IKKγ promoted aggregation andnuclear localization of mutant Htt exon-1. Moreover, in acute striatalslice cultures, inhibition of IKK activity with an N-terminallytruncated form of IKKγ, which interferes with IKK activity, blockedmutant Htt-induced toxicity in medium-sized spiny neurons (MSNs). Inaddition, blocking degradation of NF-κB inhibitors with adominant-negative ubiquitin ligase β-transducin repeat-containingprotein also reduced the toxicity of mutant Htt in MSNs.

Example 1 Mutant Htt Activates the NF-κB Pathway

This example illustrates activation of the NF-κB pathway by mutant Htt.

Materials and Methods

Cells and transgenic animals. PC12 cells, which express wild-type (WT)HDx1-enhanced green fluorescent protein (EGFP) with 25 glutamine repeats(25Qs) or mutant HDx1-EGFP with 103Qs in response to ecdysone, werekindly provided by Dr. E. Schweitzer (University of California LosAngeles). These cells were cultured on a collagen I substrate (FisherScientific, Tustin, Calif.) in DMEM with 5% horse serum, 5% fetal bovineserum, 2 mM glutamine, and standard penicillin-streptomycin antibiotics.Immortalized striatal cells from WT and HD KI mice, provided by Dr. M.E. MacDonald, were cultured as described (Gines et al., 2003). Humanembryonic kidney (HEK)-293 cells were cultured as described previously(Khoshnan et al., 2002). The HD transgenic mouse line R6/2 and aknock-in line, in which a 155 polyglutamine stretch was inserted in HDx1of mouse Htt, have been described (Davies et al., 1997; Lin et al.,2001). Colonies were established and maintained in an animal facility.

Gene Reporter and transfections. The pNF-κB-luciferase with fiveenhanced elements and the control plasmid without NF-κB binding sites,pCIS-CK luciferase, were used for gene reporter assays (Strategene, LaJolla, Calif.). To normalize for transfection efficiency betweensamples, a β-galactosidase (β-gal) construct expressed from the EF-1αpromoter (Invitrogen, Carlsbad, Calif.), was included in all genereporter assays. PC12 cells were transfected with control orNF-κB-luciferase plasmids using lipofectamine-plus. On the followingday, cells were left untreated or stimulated for 8 hr with 1 μg/mlecdysone to induce expression of HDx1. Mock or NGF (Signa, St. Louis,Mo.) was added (50 ng/ml) and incubated for an additional 6 hr. Cellswere suspended in lysis buffer on ice for 10 min and cleared bycentrifugation. Luciferase activity was measured by addition of thesubstrate (Promega, Madison, Wis.) to equal amounts of protein from eachsample. Striatal cells form WT and HD KI mice were transfected with anNF-κB reporter plus β-gal and anti-polyQ (MW2) or anti-polyproline[anti-polyP (MW7)] recombinant intrabodies (Khoshnan et al., 2002). Onthe following day, cells were starved for 2 hr and treated with 5 ng/mlrecombinant IL-1β (R&D Systems, Minneapolis, Minn.) and incubated for anadditional 6 hr. Cells were harvested and cleared lysate was used tomeasure luciferase as described above. All gene reporter assays werecorrected for transfection efficiency by normalizing the units if β-galin the extracts, using the β-gal assay (Invitrogen). β-gal values fromthe samples were divided by the β-gal value from control, 25polyQ,noninduced PC12 cells (no expression of HDx1) and multiplied by eachcorresponding luciferase reading. For striatal cells, β-gal values weredivided by the value obtained for Wt cells without any treatment.Results are shown as relative luciferase units and are representative ofat least three independent experiments. Data points are the average oftriplicate measurements.

Immunohistochemistry of tissue sections. Brains were taken fromparaformaldehyde-fixed WT and R6/2 HD transgenic mice, and cryosectionswere permeabilized in 70% methanol for 1 hr at −20° C. After blocking in3% BSA and 10% normal goat serum, slides were incubated with anti-p65(Santa Cruz Biotechnology, Santa Cruz, Calif.) and the neuronal markeranti-neuronal-specific nuclear protein (NeuN) antibodies (Chemicon),p65-positive cells were detected with a goat anti-rabbit secondaryantibody conjugated to FITC and goat anti-mouse Alexa Fluor 594(Molecular Probes). Toto-3 was used to stain nuclei. Sections wereexamined with a confocal microscope. Total neurons from 16 coronalsections containing cortical and striatal areas of four animals each ofWT and HD mice were quantified, and the average percentage of cells withnuclear p65 is presented.

Results 1. Induction of Mutant HDx1 Expression Increases Transcriptionfrom the NF-κB-Dependent Promoter Approximately Fivefold Over that ofNoninduced Cells.

To assess the ability of mutant Htt to activate the NF-κB pathway, PC12cells that express WT (25Q) or mutant (103Q) HDx1 in response toecdysone were used (FIG. 1A). These cells were transfected with areporter construct containing NF-κB enhancer element whose activation isread out as luciferase activity. The inducible nature of HDx1 expressionallows the examination of soluble mutant HDx1 function, beforemacro-aggregates become visible. Induction of mutant HDx1 expressionincreases transcription from the NF-κB-dependent promoter approximatelyfivefold over that of noninduced cells, whereas WT HDx1 has minimaleffect (FIG. 1A). These results are consistent with a recent reportdemonstrating upregulation of NF-κB expression by mutant HDx1 ininducible PC12 cells (Sugars et al., 2004).

2. Mutant Htt Influences Signal-Induced NF-κB Activation.

To examine whether mutant HDx1 also influences signal-induced NF-κBactivation, NGF, which is known to activate NF-κB in PC12 cells, wasused (Foehr et al., 2000). Mutant (but not WT) HDx1 strongly enhancesNGF-induced NF-κB dependent gene expression (FIG. 1A). Because mutantHtt has been shown to variably influence gene expression from differentpromoters, the same experiment was performed with a control plasmid thatlacks an NF-κB enhancer element. Expression of WT or mutant HDx1 doesnot influence luciferase expression from the control plasmid (FIG. 1B),suggesting that the elevated NF-κB values in FIG. 1A are mediated bymutant HDx1 expression. Under the conditions tested, most of the HDx1remains in the detergent-soluble fraction and is detectable by Westernblotting (FIG. 1A); however, the NF-κB activity diminishes as mutantHDx1 accumulates and aggregates (data not shown). Thus, soluble mutantHDx1 activates endogenous, and augments NGF-induced, NF-κB dependentgene expression. Moreover, expression of mutant HDx1 promotesdegradation of the inhibitory protein Iκ-Bα, a hallmark of NF-κBactivation. Levels of IκBα drop significantly by 3 hr and return tonormal by 7 hr after induction (FIG. 1C). These data confirm the genereporter assay results and suggest that mutant Htt-induced NF-κB ismediated by degradation of Iκ-Bα.

3. Mutant Htt Influences NF-κB Activity.

To examine whether full-length mutant Htt also influences NF-κBactivity, immortalized striatal cell lines obtained from WT and HD KImice were used (Gines et al., 2003). Using the reporter with NF-κBenhancer, no significant difference in basal NF-κB activity is observedbetween striatal cells from WT and HD KI mice (FIG. 1D) (compare Cvalues in WT and mutant). These data are consistent with a recent reportshowing that inducible expression of full-length WT or mutant Htt inPC12 cells has no effect on basal NF-κB activation (Sugars et al.,2004). Thus it was investigated whether WT and mutant cells responddifferently to NF-κB inducing agents. KI striatal cells display astronger response than WT cells to the NF-κB-inducing cytokine IL-1β(FIG. 1D).

4. Mutant Htt Mediates the Enhanced Response to IL-1β.

Recombinant single chain intrabodies, which are known to interfere withthe function of mutant HDx1 (Khoshnan et al., 2002) were used to verifythat mutant Htt mediates the enhanced response to IL-1β. Twointrabodies, MW2 targeting the expanded polyQ and MW7 recognizing thepolyP motifs of HDx1, reduce the IL-1β-mediated NF-κB activation instiratal cells from KI mice (FIG. 1D). MW7 is somewhat more potent thanMW2 at inhibiting the effects of full-length mutant Htt on NF-κB.

Activated NF-κB functions primarily in the nucleus, where it regulatesgene expression. To confirm the authenticity of the gene reporterassays, nuclear p65 binding to a consensus NF-κB oligonucleotides wasmeasured. Extracts from mutant Htt KI striatal cells show elevatednuclear p65 binding in response to IL-1β treatment when compared withthe equivalent samples from WT striatal cells (FIG. 2A). Specificity ofbinding was confirmed using a mutated consensus NF-κB oligonucleotide,which failed to show p65 binding in this assay (data not shown). Thus,cells expressing full-length mutant Htt respond more vigorously thancells with WT Htt to IL-1β-induced NF-κB nuclear localization.

5. Mutant Htt Promotes Nuclear Localization of NF-κB In Vivo.

Brain sections from 8-week-old R6/2 HD mice and age-matched controlswere stained with an antibody that recognizes the p65 subunit. p65 isconcentrated in the nucleus of a majority of the NeuN-positive neuronsin the cortex and striatum of HD mice, whereas most of the neurons ofthe age-matched WT mice contain cytoplasmic p65 (FIG. 2 B, C). Thus,mutant Htt promotes nuclear localization of NF-κB in vivo as well as incell culture.

Example 2 Mutant Htt Activates the IKK Complex

This example illustrates the activation of the IKK kinase pathway bymutant Htt.

Materials and Methods

For coimmunoprecipitation studies, PC12 cells or striatal tissue of WTand HD transgenic mice was lysed by sonication in buffer A (50 mM HEPES,pH 7.6, 250 mM NaCl, 1% Triton X-100, 2 mM MgCl₂, mM DTT, 1 mM Na₃VO₄,and 20 μm β-glycerophosphate) and a mixture of protease inhibitors(Boehringer Mannheim, Mannheim, Germany). Equal amounts of clearedextracts from each sample were incubated with a rabbit anti-IKKγ (SantaCruz Biotechnology) coupled to protein-A beads and rocked for 3 hr at 4°C. Cells were washed five times with buffer A. Immune complexes wereexamined by SDS-PAGE followed by Western blotting with monoclonalantibodies targeting Htt (Kop et al., 2001), as described in the figurelegends.

Immune complex kinase assays. To measure IKK activity,immunoprecipitated IKK complexes were examined for the ability tophosphorylate GST-Iκ-Bα in vitro. The construct for the N-terminal 61amino acids of GST-Iκ-Bα, which contains the IKK phosphorylation sites(provided by Dr. W. Greene, University of California San Francisco), wasexpressed in E. coli and purified on glutathione beads as described inthe manufacturer's instructions (Amersham Biosciences). To obtain IKKcomplexes, equal amounts of cleared striatal extracts from WT and HDanimals, or from PC12 cells induced to express WT or mutant HDx1-EGFP(all in buffer A), were incubated with a mouse anti-IKKα antibody (SantaCruz Biotechnology) coupled to protein-A beads and rocked for 3 hr at 4°C. Beads were washed five times in buffer B (50 mM HEPES, pH 7.6, 250 mMNaCl, 1 M urea, 0.1% Nonidet P-40, 6 mM EDTA, 6 mM EGTA, 1 mM DTT with20 μM β-glycerophosphate) and a mixture of protease inhibitors andequilibrated in kinase buffer (20 mM HEPES, pH 7.6, 2 mM MgCl₂, 2 mMMnCl₂, 10 μM ATP, 10 mM glycerophosphate, 10 mM NaF, 200 μM Na₃VO₄, 1 mMdithiothreitol DTT). IKK activity was evaluated in vitro with 1 μg ofpurified GST-Iκ-Bα (N-terminal 61 amino acids) in the presence of 10 μCiof [³²P]γ-ATP for 30 min at 30° C. Products were examined by SDS-PAGEfollowed by autoradiography. Duplicate samples were examined by Westernblotting using a rabbit anti-IKKγ antibody (Santa Cruz Biotechnology).

Results 1. The IKK Complex is Activated by Mutant HDx1 Expression.

Signal-induced phosphorylation of serine residues 32 and 36 of IκBα,which is mediated by the IKK complex, is essential for theubiquitination and proteosome-mediated degradatin of IκBα (Ghosh andKarin, 2002). Immunoprecipitated endogenous IKK complexes from extractsof control or PC12 cells induced to express mutant HDx1 were analyzedfor their kinase activity by measuring phosphorylation of therecombinant substrate, GST-IκBα. The kinase activity of IKK from cellsexpressing mutual HDx1 is significantly higher than that fromnon-HDx1-induced cells (FIG. 3A). As expected, NGF treatment alsoactivates the IKK complex, and consistent with the gene reporter assayresults, kinase activity appears to be elevated somewhat further whenboth NGF and HDx1 are present (4.1-fold compared with a 2.4- and3.2-fold in the ecdysone and NGF-treated cells, respectively).Expression of WT HDx1 has no effect on IKK activity (not shown).

2. IKK Activation by Mutant HDx1 Occurs In Vivo.

Two lines of HD transgenic mice, R6/2 (Davies et al., 1997) and a mutantHtt KI (Lin et al., 2001) were used to investigate whether IKKactivation by mutant HDx1 also occurs in vivo. IKK complexesimmunoprecipitated from striatal or cortical extracts of WT and HD micewere assayed in vitro for IKK activity. Consistent with the PC12 cellresults, IKK activity is higher in complexes isolated from striatal(9-fold) as well as cortical (3.8-fold) extracts of 2-month-old R6/2mice compared with age-matched WT controls (FIG. 3B). With KI HD mice, asignificant increase in IKK activity is detected in the striatum at 1year of age (4.8-fold higher than WT) (FIG. 3C).

3. HDx1 Associates with IKK.

Complexes isolated with an anti-IKK antibody from PC12 cells wereexamined for the presence of HDx1 by Western blotting. Soluble mutantHDx1 coprecipitates with IKK, whereas minimal WT HDx1 is observed (FIG.3D). Thus, IKK physically forms a complex with mutant HDx1. Thisinteraction also occurs in the brain of HD mice. IKK complexes isolatedfrom brain extracts of R6/2 mice contain soluble HDx1, which isrecognized by anti-Htt antibody (FIG. 3E).

Example 3 Mutant Htt Interacts with IKKγ

This example illustrates the interaction of mutant Htt with IKKγ.

Materials and Methods

Protein-protein interaction. In vitro binding of IKKγ to mutant HDx1 wasperformed using glutathione S-transferase (GST)-pull down assays.GST-HDx1 with 20 or 51 polyQs, with and without polyproline domains, wasobtained from Dr. E. Wanker (Max Plank Institute, Berlin, Germany).These constructs were expressed in Escherichia coli and purified withglutathione beads (Amersham Biosciences, Piscataway, N.J.) according tomanufacturer's instructions. IKKγ constructs were transcribed andtranslated in rabbit reticulocytes in the presence of [³⁵S] methionine(Promega). Briefly, 5 μg of GST-HDx1 bound to beads was incubated with10 μl of each labeled in vitro-translated IKKγ produce in 500 μl ofTris-based buffer containing 10% glycerol and 6 mM DTT and rocked atroom temperature for 2 hr. Beads were washed five times in the samebuffer. Bound IKKγ was examined by SDS-PAGE, followed byautoradiography. Recombinant anti-HDx1 intrabodies (Khoshnan et al.,2002) were expressed and examined similarly fro binding to GST-HDx1. Forcompetition assays, 10-fold excess in vitro-translated intrabodies wereadded to GST-HDx1 plus deleted C terminus IKKγ and processed as above.

Results 1. The Binding of Mutant HDx1 to the IKK Complex is Mediated byIKKγ.

The activity of the IKK complex is regulated by IKKγ, a glutamine-richnonkinase subunit of the complex (Rothwarf et al., 1998); Ghosh andKarin, 2002). A homolog of IKKγ that participates in tumor necrosisfactor-α receptor signaling interacts with Htt in a yeast two-hybridassay (Hattula and Paranen, 2000). A GST pull-down assay using HDx1fused to GST and [³⁵S] methionine-labeled IKKγ was used to determinewhether the binding of mutant HDx1 to the IKK complex is mediated byIKKγ. The stringency of the assay was such that there was no binding toGST alone. In vitro translated-IKKγ preferentially binds to GST-HDx1containing expanded polyQ (FIG. 4A). Although deletion of the C-terminal119 amino acids of IKKγ (DC-IKKγ) has no apparent effect on thisbinding, removal of the first 134 amino acids (DN-IKKγ) diminishesbinding to HDx1 (FIG. 4A). Interestingly, mutant HDx1 without its polyPdomains does not bind efficiently to Ikkγ (FIG. 4B). Although it is notpossible to rule out that the absence of proline may also the solubilityof mutant HDx1 and thus artifactually reduce binding to IKKγ, theimportance of the polyP domains is further supported by antibodyexperiments. Competition assays using in vitro-translated, anti-Httrecombinant intrabodies have been performed (Khoshnan et al., 2002).Coincubation of DC-IKKγ plus mutant HDx1 with 10-fold excess ofintrabodies specific for polyQ (MW2) or polyP (MW7) competes withbinding of IKKγ to HDx1 (FIG. 4B). Moreover, these intrabodies inhibitthe HDx1-induced NF-κB-dependent gene expression in PC12 cells (FIG.4C). Although anti-polyQ intrabody has a modest inhibitory effect,anti-polyP (MW7) significantly minimizes mutant HDx1-induced NF-κBactivation. Thus, IKKγ binding to mutant HDx1 requires the polyQexpansion and is influenced by the polyP domains.

Example 4 Inhibition of the NF-kB Pathway Reduces HDx1-Induced Toxicityin Cell Culture and Brain Slices

This example illustrates the protection against mutant Htt toxicity inMSNs by inhibition of the NFκB pathway.

Materials and Methods

Gene Reporter and transfections. To examine the effects of ΔF-βTrCP(obtained from Dr. R. Deshaies (California Institute of Technology) andoriginally provided by Spencer et al. (1999)) and deleted N terminus(DN)-IKKγ on mutant HDx1-induced NF-κB, PC12 cells were transfected withΔF-βTrCP or DN-IKKγ plus β-gal and NF-κB reporter in six-well plates.Induction of HDx1 with ecdysone and NGF treatment was as describedabove. Equal amounts of each sample were used to measure luciferaseactivity. Binding of NF-κB to its consensus oligonucleotide site wasassayed in striatal neurons with the BD TransFactor Kit specific for thep65 subunit following the manufacturer's instructions (BD Biosciences,Mountain View, Calif.). Briefly, striatal cell lines from WT and HD K1mice were starved for 2 hr and treated with 6 ng/ml IL-1β for 15 or 30min. Nuclear extracts were obtained using commercial reagents (BDBiosciences). Protein concentration was determined by the BCA method(Pierce, Rockford, Ill.). Equal amounts of each nuclear fraction wereadded to wells coated with an NF-κB consensus or mutated controloligonucleotide and incubated at room temperature for 1 hr. Wells werewashed extensively and incubated with primary rabbit antibody specificfor the p65 subunit of NF-κB. Bound p65 was detected with a goatanti-rabbit antibody conjugated to horseradish peroxidase. Afteraddition of the substrate 3,3-5,5-tetramethylbenzidine, colordevelopment was measured at 655 nm using an ELISA plate reader. Sampleswere done in triplicate, and the results shown are representative ofthree independent experiments.

Transfection of HEK-293 cells with mutant HDx1-EGFP alone (Kasantsev etal., 1999) or with one of the following constructs, full-length (F), DN,and deleted C terminus (DC) of IKKγ (provided by Dr. E. Zandi,University of Southern California) Rothwarf et al., 1998) or ΔF-βTrCP,was done using lipofectamine-plus reagents (Invitrogen). Forhistochemistry, cultured cells were fixed in 4% paraformaldehyde for 30min, permeabilized with 70% methanol for 1 hr., and washed with PBS.Cells were stained with anti-HA for IKKγ or anti-myc for ΔF-βTrCP (CellSignaling, Beverly, Mass.) followed by a goat anti-mouse antibodyconjugated by Alexa 594 (Molecular Probes, Eugene, Oreg.). Toto-3, adimeric cyanine, was used to stain nuclei (Molecular Probes). Cells werewashed in PBS, mounted on microscope slides, and examined with aconfocal microscope. Mutant HDx1 toxicity was assessed by counting thenumber of condensed GFP-positive bodies, which are remnants ofterminated deoxynucleotidyl transferase-mediated biotinylated UTP nickendlabeling (TUNEL)-positive (TUNEL⁺) cells (Khoshnan et al., 2002),using a florescence microscope. For TUNEL staining, transfected cellsgrown on coverslips were air dried and fixed 16 hr after transfection asabove and permeabilized in ethanol/acetic acid (2:1). Apoptotic cellswere labeled with terminal deoxynucleotidyl transferase usingdigoxigenin-labeled nucleotides and detected by anti-digoxigeninantibody and rhodamine-conjugated secondary according to theinstructions provided by the manufacturer (Chemicon, Temecula, Calif.).

Brain slice preparation. All animal experiments were performed inaccordance with the Institutional Animal Care and Use Committee and DukeUniversity Medical Center Animal Guidelines. Postnatal day 7 (P7) CDSprague Dawley rats (Charles River Laboratory, Raleigh, N.C.) weredecapitated, and the brains were surgically removed and placed inice-cold Neurobasal medium (Invitrogen). The tissue was fixed to achilled, stainless steel Vibratome stage using cyanoacrylate glue(Krazy-Glue) and covered with Neurobasal medium. Coronal brain slices(250 μm thick) were cut by Vibratome (VT1000S; Leica, Nusslock Germany)as described previously (Edgerton and Reinhart, 2003). Brain slices werekept at 37° C. in 5.0% CO₂ for 1 hr.

Bolistic transfection. Gold particles (1.6 μm gold microcarriers;BioRad, Hercules, Calif.) were used as DNA carriers for transfection asdescribed previously (Lo et al., 1994). Briefly, gold particles weresonicated in 0.05 M spermidine in the presence of plasmid DNA. Thegold-DNA mixture was washed three times in 100% ethanol before beingloaded into Helios plastic cartridges (Bio-Rad) according tomanufacturer's instructions. Slices were transfected using a Bio-RadHelios gun with a cyan fluorescent protein (CFP)-tagged Htt fragment,together with yellow fluorescent protein (YFP) as a morphometric marker.In a number of experiments, one of the IKKγ constructs was alsocotransfected with the Htt-construct and YFP. CFP-tagged Htt fragmentswere exon-1-N-terminal fragments containing either a short-Q (Q23) or along-Q (Q148) polyQ domain.

Brain slice neurodegeneration assay. For each condition, transfectionswere performed on twelve brain slices per experiment. Protein expressionand neurodegeneration (loss of processes, shriveling of the soma) wereassay 2-7 d after transfection using a Leica fluorescence microscopewith appropriate filters for YFP and CFP. The total area of the striatumwas identified, and transfected MSNs were identified by theirmorphology. CFP fluorescence was used to determine expression of Httfragments and their aggregation into macro-inclusions. Each experimentinvolved three to twelve transfected brain slices per condition, and thedata are means from six independent experiments for each condition.

Results 1. IKKγ can Modify the Toxicity of Mutant HDx1.

Confocal microscope examination of HEK-293 cells expressing mutant HDx1and IKKγ at an early time point before widespread toxicity shows thatfull-length IKKγ promotes aggregation of mutant HDx1 (FIG. 5A).Intracellular, microscopically visible aggregates are apparent, andimportantly, some of these aggregates localize in the nucleus oftransfected cells. C-terminally truncated IKKγ, which also binds tomutant HDx1 (FIG. 4A), has a similar effect (data not shown). On theother hand, DN-IKKγ, which does not bind to mutant HDx1, has no apparenteffect on HDx1 aggregation (FIG. 5A). Thus, binding to IKKγ appears tohave little effect on toxicity at 48 hr after transfection, however,because the overall number of calls killed by mutant HDx1 does notappear to change (FIG. 5A). Toxicity was assessed by counting the numberof condensed GFP⁺ bodies at 48 hr after transfection, which are remnantsof dead cells (Khoshnan et al., 2002) (see FIG. 7B). Importantly,DN-IKKγ strongly reduces the toxicity of mutant HDx1 (FIG. 5A, graph).The effect of DN-IKKγ on mutant HDx1 toxicity was confirmed by TUNELassay. Compared with cultures with or without full IKKγ, the number ofTUNEL⁺ cells is significantly reduced in cells cotransfected withDN-IKKγ and mutant HDx1 (FIG. 5B). DN-IKKγ lacks the binding domainessential for interaction with IKKα and -β and interferences with IKKactivity (May et al., 2000; Poyet et al., 2000). DN-IKKγ also blocksbasal and NGF-induced NF-κB-dependent gene expression by mutant HDx1 ininducible PC12 cells (FIG. 5C). Therefore, inhibition of mutant HDx1toxicity by DN-IKKγ may be mediated through blockage of NF-κB pathway.

2. DN-IKKγ Reduces the Neurotoxicity of Mutant HDx1 In Vivo.

A novel, acute, P7 rat brain slice assay was used to assess the effectsof IKKγ in vivo. A biolistic method is used to cotransfect neurons withthree constructs HDx1-CFP to visualize HDx1 expression and associatedneurodegeneration, YFP to monitor the full morphology of transfectedcells, and IKKγ or DN-IKKγ. Individual transfected MSNs are identifiedby location and morphology and monitored daily for 7 d aftertransfection. Neuronal viability was quantified by counting the numberof CFP⁺ neurons in each slide at 7 d. Expression of mutant HDx1 aftertransfection and to cell death within 4-7 d. YFP apoptotic bodies areseen initially, followed by complete loss of YFP fluorescence. Incontrast, neurons transfected with WT HDx1 do not contain Htt inclusionsand retain their normal dendritic structure (FIG. 6A). Coexpression offull-length IKKγ or DN-IKKγ has no obvious effect on MSNs expressing WTHDx1 (FIG. 6A, B). Furthermore, expression of IKKγ has no significanteffect on neurodegeneration induced by expression of mutant HDx1 (FIG.6A, B); however, the coexpression of DN-IKKγ with mutant HDx1 issignificantly neuroprotective (FIG. 6A, B). The neuroprotective functionof DN-IKKγ is essentially independent of aggregate formation by mutantHtt, because rescued neurons still contain Htt macro-aggregates (FIG.6A). These brain slice results indicate that DN-IKKγ also reduces theneurotoxicity of mutant HDx1 in MSNs residing in a more intact,three-dimensional setting.

3. Blocking Degradation of Iκ-B can Reduce the Toxicity of Mutant HDx1.

The E3-ubiquitin ligase βTrCP specifically promotes degradation of Iκ-Bα(Spencer et al., 1999). A dominant-negative form of βTrCP (ΔF-βTrCP),which has been shown to block degradation of phosphorylated Iκ-BS, wascotransfected with the NF-κB reporter in PC12 cells. Expression ofΔF-βTrCP abolishes basal and mutant HDx1-induced NF-κB activity (FIG.7A). As predicted ΔF-βTrCP expression also blocks the toxicity of mutantHDx1 in cultured cells (FIG. 7B, C). Cells expressing ΔF-βTrCP andmutant HDx1 remain intact, whereas cells transfected with mutant HDx1and control vector become condensed and eventually detach from thecultured dish (FIG. 7B, arrow). Moreover, expression of ΔF-βTrCP in thebrain slice assays significantly reduces the toxicity of mutant HDx1 inMSNs (FIG. 7D). WT-βTrCP does not influence the toxicity of mutant HDx1in MSNs (FIG. 7D). Collectively these data show that blocking IKKactivity or degradation of Iκ-B, both of which result in inhibition ofNF-κB, can reduce the toxicity of mutant HDx1.

Example 5 Inhibition of IKK by a Small Molecule Inhibitor Reduces HttToxicity in Cells

This example illustrates the protection of cells from the toxicity ofmutant Htt by an IKK inhibitor.

To examine the effects of a small molecule IKK inhibitor on mutant Htttoxicity, PC12 cells expressing mutant HDx1 are treated with herbimycinor another known IKK inhibitor plus β-gal and NF-κB reporter in six-wellplates, essentially as described above in Example 4. Herbimycin isobserved to reduce NF-κB expression and blocks the toxicity for mutantHDx1 in the cultured cells.

HEK-293 cells are transfected with mutant HDx1-EGFP, treated withherbimycin treatment or another IKK inhibitor, and analyzed forapoptosis using TUNEL staining as described above in Example 4. Thenumber of TUNEL stained cells is significantly reduced in cells treatedwith the IKK inhibitor.

P7 rat brain slices are transfected with mutant HDx1 as described abovein Example 4 and treated with herbimycin or another known IKK inhibitor.The IKK inhibitor significantly reduces toxicity of mutant HDx1 in MSNs.

Example 6 Identification of Therapeutics for the Treatment of HD

This example illustrates the identification of compounds that can beused to treat or prevent HD.

Compounds to be tested for the potential to be effective therapeuticsfor HD are provided. As discussed above, the compounds may be, withoutlimitation, small molecules, peptides, polypeptides, or antibodies. Insome embodiments the compounds are initially screened for their abilityto interact with IKK. Compounds that interact with IKK are then testedfor their ability to inhibit IKK activity, for example in an immunecomplex kinase assay or a gene reporter assay. Compounds that are ableto inhibit IKK activity are then tested for their ability to protectcells from the toxic effects of mutant Htt, for example as describedabove in Example 5.

In other embodiments, compounds are provided that are related to knownIKK inhibitors. For example, they may be structurally related. Thesecompounds are first tested for their ability to inhibit IKK activityand, if they appear to be candidate IKK inhibitors, are then tested fortheir ability to protect cells from the toxic effects of mutant Htt.

In still other embodiments, compounds are tested directly in assays oftheir ability to protect cells from the toxic effects of mutant Htt,without first directly testing their ability to inhibit IKK.

Compounds that show some efficacy in protecting cells from the toxiceffects of mutant Htt may then be tested for their efficacy and toxicityin animal models of HD and in clinical trials on human patients.

Example 7 Treatment of Huntington's Disease

This example illustrates the treatment of a patient suffering from HD.

A patient suffering from or at risk of developing HD is identified andadministered an effective amount of a composition comprising an IKKinhibitor. A typical daily dose for an IKK inhibitor of the presentinvention might range from about 0.01 μg/kg to about 1 mg/kg of patientbody weight or more per day, depending on the factors mentioned above,preferably about 10 μg/kg/day to 100 10 μg/kg/day. The appropriatedosage and treatment regimen can be readily determined by the skilledartisan based on a number of factors including the nature of the IKKinhibitor, the route of administration and the patient's disease state.HD treatment efficacy is evaluated by observing delay or slowing ofdisease progression, amelioration or palliation of the disease state,and remission.

Although the present invention has been described in detail above, itwill be understood by the skilled artisan that various modifications canbe made without departing from the spirit of the invention. Accordingly,the invention is limited only by the following claims. All citedpatents, patent applications and publications referred to in thisapplication are herein incorporated by reference in their entirety.

1. A method for protecting cells from the toxicity of mutant Htt,comprising inhibiting I-κB kinase (IKK) activity in said cells.
 2. Themethod of claim 1, wherein inhibiting comprises contacting said cellswith an IKK inhibitor.
 3. The method of claim 2, wherein said IKKinhibitor binds to IKKβ.
 4. The method of claim 2, wherein said IKKinhibitor blocks interaction of IKKγ with IKKβ and IKKα.
 5. The methodof claim 2, wherein said IKK inhibitor blocks IKKβ activation.
 6. Themethod of claim 2, wherein said IKK inhibitor is selected from the groupconsisting of a small molecule, a peptide and an antibody.
 7. The methodof claim 6, wherein said IKK inhibitor is a small molecule.
 8. Themethod of claim 7, wherein said small molecule is selected from thegroup consisting of herbimycin, sodium salicylate, retinoid-relatedcompounds, and cyclopentenone prostaglandins.
 9. The method of claim 6,wherein the IKK inhibitor is a NEMO binding peptide.
 10. The method ofclaim 6, wherein said IKK inhibitor is an antibody.
 11. The method ofclaim 1, wherein the cells are from a patient.
 12. The method of claim11, wherein the patient is a human.
 13. The method of claim 1, whereinsaid cells are neurons.
 14. The method of claim 13, wherein said neuronsare medium-sized spiny neurons (MSNs).
 15. A method for treatingHuntington's disease, comprising administering to a patient in need ofsuch treatment an effective amount of an I-κB kinase (IKK) inhibitor.16. The method of claim 15, wherein said IKK inhibitor binds to IKKβ.17. The method of claim 15, wherein said IKK inhibitor blocksinteraction of IKKγ with IKKβ and IKKα.
 18. The method of claim 15,wherein said IKK inhibitor blocks IKKβ activation.
 19. The method ofclaim 15, wherein said IKK inhibitor is selected from the groupconsisting of a small molecule, a peptide and an antibody.
 20. Themethod of claim 19, wherein said IKK inhibitor is a small molecule. 21.The method of claim 20, wherein said small molecule is selected from thegroup consisting of herbimycin, sodium salicylate, retinoid-relatedcompounds, and cyclopentenone prostaglandins.
 22. The method of claim19, wherein the IKK inhibitor is a NEMO binding peptide.
 23. The methodof claim 19, wherein said IKK inhibitor is an antibody.
 24. The methodof claim 15, wherein the patient is a mammalian patient.
 25. The methodof claim 24, wherein the mammalian patient is human.
 26. The method ofclaim 19, wherein the IKK inhibitor is delivered intracranially.
 27. Themethod of claim 26, wherein the IKK inhibitor is injected directly intobrain tissue.
 28. The method of claim 26, wherein the IKK inhibitor isinjected into the cerebrospinal fluid.
 29. The method of claim 15,wherein said IKK inhibitor blocks mutant Htt-induced toxicity inneurons.
 30. The method of claim 29, wherein said neurons aremedium-sized spiny neurons (MSNs).
 31. A method of identifying compoundsfor treating Huntington's disease comprising: providing one or morecompounds to be tested; identifying which of said compounds are I-κBkinase (IKK) inhibitors; and testing the compounds identified as IKKinhibitors for their ability to protect cells from the toxicity ofmutant Htt.
 32. The method of claim 31, wherein the compounds areselected from the group consisting of small molecules, peptides andantibodies.
 33. The method of claim 31, wherein identifying compoundsthat are IKK inhibitors comprises testing compounds for their ability toinhibit the ability of IKK to phosphorylate a substrate.
 34. The methodof claim 33, wherein compounds are tested for their ability to inhibitIKK phosphorylation in an immune complex kinase assay.
 35. The method ofclaim 31, wherein identifying compounds that are IKK inhibitorscomprises identifying compounds that bind IKK.
 36. The method of claim35, additionally comprising testing compounds that bind IKK for theirability to inhibit NF-κB activity.
 37. The method of claim 36, whereinthe compounds are tested for their ability to inhibit NF-κB activity ina gene reporter assay.
 38. The method of claim 31, wherein the cells areneurons.
 39. The method of claim 38, wherein compounds are tested fortheir ability to protect neurons from the toxicity of mutant Htt in anin vivo brain slice assay.